Insulin is a low molecular weight (ca. 5800 Da) polypeptide hormone produced by the pancreas. It is responsible for regulating the metabolism of carbohydrate and blood glucose levels. The human form is a peptide composed of 51 amino acids, a 21-residue A-chain and a 30-residue B-chain linked by two disulfide bonds. A determination of circulating insulin in serum or plasma is of intrinsic value in the clinical diagnosis and classification of various types of diabetes and related diseases and doping control in athletes.
During the past decade, a variety of detection methods, including those based on radioimmunoassay, mass spectrometry, fluorescence spectrometry and surface plasmon resonance have been developed for the determination of insulin. These methods are, though, either impractical or insensitive and are not translatable to a point-of-care format.
With an increase in the incidence of diabetes, the development of an ultrasensitive, cheap, simple and automated diagnostic test would be of considerable value. In attempting to meet these requirements, electrochemical analyses have received attention. Though amperometric sensors based on the oxidation of insulin have been reported, these operate at high potentials, and accordingly suffer from ascorbic acid and uric acid interference. They are also comparatively insensitive; the blood content of insulin is normally below 80 pM between meals, much lower than the detection limits of most of electrochemical sensors reported to date.
Electrochemical impedance spectroscopy (EIS) is a technique that is capable of sensitively monitoring the changes in capacitance or charge-transfer resistance associated with the specific binding of certain materials to a suitably modified electrode surface. Some immunoassays based on EIS have been demonstrated for the detection of relatively large protein molecules (MW>20 kD). However, EIS has generally not been applied to the analysis of smaller molecules, such as low molecular weight polypeptides, where the impact on impedance resulting from interfacial binding would be expected to be much lower than in the case of large proteins; this is a particularly significant consideration in the context of complex biological fluids containing very low concentrations of the molecules of interest.